Abstract

Gene therapy, the ability to treat a disease at the level of nucleic acid, has journeyed from science fiction, to hard lessons
learned from early clinical trials, to improved technologies with efficacy in patients for several diseases. Adeno‐associated
virus (AAV) vectors are currently a leader for direct in vivo gene therapy. To date, AAV is safe in patients, with clinical benefit in trials to treat blindness, hemophilia, and a lipid
disorder, with many more trials underway. Despite this remarkable progress, barriers exist for AAV vectors to be effective
gene transfer vehicles in all organ/cell targets, as well as patient subpopulations. Extracellular vesicles (EVs, e.g., exosomes,
microvesicles) are natural lipid particles released by many cell types. They have been reported to mediate cell to cell communication
via transferred contents including proteins, nucleic acids, and metabolites. These properties of EV attracted our attention
to help solve certain gene transfer issues encountered by AAV vectors. We made the initial discovery that a subpopulation
of AAV vectors isolated from media directly interacted with EVs [referred to as exosome‐associated AAV (exo‐AAV)]. In following
reports, we have demonstrated that exo‐AAV has advantages over the conventional AAV vector in areas such as anti‐AAV antibody
evasion and transduction of cells of the eye and cochlea in preclinical models. The work of others using EVs as therapeutics
as well as our continued development of the exo‐AAV platform may advance the field towards useful clinical applications.

Images

(a) BALB/c mice were injected i.v. with 5 × 1010 g.c. of either standard or exo‐AAV‐FLuc (both AAV8 and AAV9 capsids tested). Representative images of bioluminescence intensity of head region in mice 7 days post‐injection. (b) Bioluminescence signal quantitation from (a). (c) Exo‐AAV cross a BBB model. bEnd.3 cells, a cell line generated from mouse cortical endothelial cells, were seeded on cell culture inserts and grown to confluence to establish an in vitroBBB model. Standard AAV or exo‐AAV (1.6 × 1010 g.c.) were suspended in media and dispensed into the upper chamber (apical side of cell culture inserts), while vector‐free media was added to the lower chamber (below the inserts). After 24 h, media was collected from the lower chamber to determine the number of AAV genomes present in the media by quantitative polymerase chain reaction (qPCR). Data are presented as the mean of total AAV g.c. below the cell culture insert ± SDN = 4 transwell assays per group; t‐test between standard AAV and exo‐AAV for each serotype; *, P AAV9 and exo‐AAV9. Double immunostainings were performed to determine the main neural cell types transduced by both exo‐AAV9 and AAV9 (white arrows). Representative images of GFP‐positive astrocytes and neurons, respectively, identified by the markers GS and NeuN, in the cortex of injected mice. Scale bar: 50 µm. (Reprinted with permission from Ref Copyright 2016 Nature Publishing Group)

Conventional AAV9 or ev‐AAV9 (AKA exo‐AAV) isolated from media by different g‐forces (20,000 × g or 100,000 × g) were compared for evasion of anti‐AAV antibodies in IVIg. (a) An in vitro neutralization assay was performed for each vector. Greater resistance to antibodies was observed for exo‐AAV compared to conventional AAV. (b) Mice were injected with phosphate‐buffered saline (PBS) or 0.5 mg IVIg/mouse and 24 h later challenged i.v. with 1010 genome copies (g.c.) of either conventional AAV9‐FLuc or 100K × g exo‐AAV9‐FLuc. Fourteen days later, mice were imaged for FLuc expression by bioluminescence imaging. Bioluminescent images of the head region are shown. Note the scale differences between conventional and ev‐AAV. (c) Photon flux of head region for conventional AAV9‐FLuc or 100K × g exo‐AAV9‐FLuc with PBS or IVIg pretreatment. (d) Residual transduction in the IVIg groups relative to PBS groups. *, P Copyright 2014 Elsevier)

Advantages of exo‐AAV over conventional AAV. (a) Increased uptake by cells not susceptible to efficient transduction by conventional AAV. If a cell expresses AAV receptors (red receptors), transduction may occur (green cell indicates efficient transduction). If these receptors are absent on a nonsusceptible cell, transduction is inefficient (blue cell). As exosomes can enter cells through multiple receptors (yellow and green receptors), exo‐AAV may increase transduction of previously nonsusceptible cells. (b) Exo‐AAV has an increased ability to transduce cells even in the presence of anti‐AAV antibodies (indicated in green). (c) Engineering ligands and transmembrane receptors onto the exosome surface can increase delivery to target tissues. Transmembrane ligands indicated as magenta structures on exosome surface. (d) Packaging two different vectors (indicated by a red and a green capsid) into exosomes may increase the efficiency of two‐component vector systems.

Efficient gene delivery to the inner ear with exo‐AAV for gene therapy of hearing and balance disorders. (a) CD1 mice were injected at P1 with 5 × 109 g.c. of conventional AAV1 or exo‐AAV1 through the round window membrane (RWM). Efficient outer hair cell (OHC) transduction by exo‐AAV1 compared to conventional AAV1. The insets in the lower panels show the outlined region of the main panel at the same magnification but with higher brightness. All image post‐processing was done identically between AAV1 and exo‐AAV1. Scale bars are 20 µm. (b) Percentage of GFP‐positive hair cells in four regions of the cochlea (base, midbase, midapex, apex), transduced with conventional AAV1 or exo‐AAV1. Mean ± SEM, **, P P U‐test between AAV1 and exo‐AAV1. R2 is the coefficient of determination for the average values in each region; it tests whether there is a correlation between the location and transduction efficiency. (c)–(g) Rescue of hearing and balance in the Lhfpl5 knockout mouse. (c) RWM injection of exo‐AAV1‐CBA‐HA‐Lhfpl5 through the round window at P1 restores FM1‐43 (a dye which is only taken up by functional HCs) loading in inner hair cells (IHCs) and OHCs (7 days after injection; P6 + 2) of Lhfpl5−/− mice. Scale bar, 20 µm. (d) High magnification images show anti‐HA staining in the bundles of an IHC and an OHC. Anti‐HA staining is detectable at the tips of all rows of stereocilia. Scale bar, 2 µm. (e) Auditory brain stem response (ABR) waveforms at 8 kHz from heterozygous, uninjected Lhfpl5−/− and exo‐AAV1‐CBA‐HA‐Lhfpl5‐injected Lhfpl5−/− animals. Sound pressure level is shown in dB. ABR was recorded at 4 weeks post‐injection. (f) Lhfpl5−/− knockout mice injected with exo‐AAV1‐CBA‐HA‐Lhfpl5 through the RWM at P1. Solid black circles represent uninjected Lhfpl5−/− ears and show no detectable ABR at any sound pressure level. Open circles show heterozygous control thresholds at this age. Colored symbols represent nine individual animals that showed some level of rescue after exo‐AAV1‐CBA‐HA‐Lhfpl5 injection. (g) Behavior tests to monitor movement abnormalities in treated and untreated mice. Left: head tossing was quantified by blinded investigators. Head tossing in exo‐AAV1‐CBA‐HA‐Lhfpl5‐injected knockouts was less than in uninjected knockouts, and not detected at all in 5 out of 12 injected animals (green circles). Right: circling was also decreased in exo‐AAV1‐CBA‐HA‐Lhfpl5‐injected knockouts. Circling was quantified using EthoVision XT software (Noldus), and full 360° turns were identified as a circle. *, P P U‐test. Mean ± SD. (Reprinted with permission from Ref Copyright 2017 Cell Press)

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started out in mechanical engineering and became interested in nanotechnology with his studies on nanomechanics and nanofluidics. His research work and involvement with setting up some of the premier nano centers and alliances in the world, bringing together universities, hospitals, and federal agencies, showcases interdisciplinarity at work.